A printed Sequence Listing accompanies this application, and also has been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette.
1. Field of the Invention
The present invention is broadly concerned with compositions of two or more peptides effective in inhibiting the binding of proteins, and corresponding methods of use. More particularly, the preferred form of the invention relates to peptide compositions which inhibit the binding of leukocyte function-associated antigen (LFA-1) and intercellular adhesion molecule (ICAM-1); these peptide compositions can be used to treat disease states such as rejection of transplanted organs, allergies, and autoimmune diseases.
2. Description of the Prior Art
Many biological phenomena involve the mutual recognition of proteins. For example, it has been known for decades that antibodies bind to antigens in order to protect the body against foreign substances (Carayannopoulos et al., 1993, Imnunoglobulins, Structure and Function, In: Fundamental Immunology, Paul, W. E., Ed., pp. 283-314, Raven Press). Also, Ras and Raf-1 are proto-oncoproteins that transduce growth and differentiation signals initiated: by tyrosine kinases; ras binds to Raf-1 and thereby inhibits Ras-GAP activity (Zhang et al., 1993, nature 364:308-313). Yeast Cdc7 protein kinase and Dbf protein are both required for the initiation of DNA replication; these proteins bind to each other, and it is thought that Dbf4 is specific for the activation of Cdc7 kinase (Jackson et al., 1993, Mol. Cell Biol. 13:2899-2908). Yeast GAL4 protein consists of two protein subunits which, when bound together, activate the genes encoding enzymes of galactose utilization (Fields et al., 1989, Nature 340:245-246).
It is also widely known that the contact sites within proteins that bind to one another are noncontinuous domains of amino acids. These contact sites can be found in different subunits of a protein as in the case of the heavy and light chains of antibodies (Carayannopoulos et al., 1993; Perutz, 1992, Protein Structure, New Approaches to Disease and Therapy, pp. 41-76, W. H. Freeman and Co.). Alternatively, these contact sites can be found in different areas of the same subunit as in the case of the xcex1 subunit of LFA-1 (Edwards et al., 1995, J Biol Chem. 270:12635-12640; Stanley et al., 1994, EMBO 13: 1790-1798).
Many autoimmune diseases occur when T-cells of an organism recognize and react to xe2x80x9cselfxe2x80x9d proteins. This recognition occurs when specific proteins on the surface of the T-cells bind to the corresponding self proteins. This type of reaction results in rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis. Allograft rejection also results from T-cell attack.
Initiation of an immune response to an antigen involves interaction of a small subset of T-cells with the antigen, followed by activation and proliferation of those T-cell clones. Complete T-cell activation requires two signals: (1) interaction of the T-cell receptor with an appropriate MHC-antigen complex, and (2) a second signal provided by adhesion molecules. The second signal may be provided by binding of the adhesion receptor, LFA-1 (CD 11a and CD 18), to one of its counter-receptors such as ICAM-1 (CD54) (Staunton et al., 1990, Cell 61:243-254). If the second signal is blocked, the antigen-specific T-cells are induced to die by apoptosis or to enter a state of cellular anergy. Blockage of this interaction by monoclonal antibodies to LFA-1 and ICAM-1 results in increased survival time for mice receiving heart allografts (Isobe et al., 1992, Science 255:1125-1127).
Previous studies have shown that peptide fragments from ICAM-1 can inhibit T-cell-endothelial adherence (Ross et al., 1992, J Biol. Chem. 267:8537-8543), HIV-1 replication in MT-2 cells (Fecondo et al., 1993 Aids Research and Human Retroviruses 9:733-740), homotypic adhesion of Raji cells, and cytotoxic cell-mediated killing of K562 cells. Peptides from the sequence of the xcex1 subunit of LFA-1 have also been shown to inhibit binding of T-cells to ICAM-1 (Stanley et al., 1994, EMBO 13:1790-1798). Additionally, short-chain peptides derived from active sites of ICAM-1 and LFA-1 also inhibit these types of interactions (Benedict et al., 1995, International Publication No. WO 95/28170, the teachings of which are incorporated by reference herein). However, there are no reports in the prior art of peptide compositions which are effective in inducing immune tolerance in an organism.
The present invention is predicated upon the idea that compositions of short-chain peptides can inhibit the binding of one protein (a first protein) to another protein (a second protein). The mutual binding of a pair of proteins is responsible for signal transductions occurring in many biological processes. In the case of the immune response, inhibition of such protein binding can result in induction of immune tolerance. Therefore, the peptide compositions of the present invention can be used, for example, as a treatment for disease states such as rejection of transplanted organs, allergies, and autoimmune diseases (e.g., rheumatoid arthritis, insulin-dependent diabetes mellitus, and multiple sclerosis).
Specifically, the present invention is directed to peptide compositions, and methods of using these compositions, wherein at least one peptide binds to the first protein, and at least one peptide binds to the second protein, whereby the first protein is inhibited from binding to the second protein. Preferably, the first protein is an integrin (e.g., the xcex1 and xcex2 subunits of LFA-1) while the second protein is an integrin-binding protein (e.g., ICAM-1).
If the protein system is the LFA-1/ICAM-1 system, each peptide which binds to LFA-1 is derived from ICAM-1, while each peptide which binds to ICAM-1 is derived from LFA-1. Ideally, each ICAM-1-based peptide contains a sequence present in a sequence selected from the group consisting of Sequence ID Nos. 1-14, contains a sequence selected from the group consisting of Sequence ID Nos. 1-14, or has a sequence selected from the group consisting of Sequence ID Nos. 1-14; furthermore, each LFA-1-based peptide contains a sequence present in a sequence selected from the group consisting of Sequence ID Nos. 15-35, contains a sequence selected from the group consisting of Sequence ID Nos. 15-35, or has a sequence selected from the group consisting of Sequence ID Nos. 11-35.
Advantageously, each peptide is not immunogenic, and has a molecular weight under 20 kilodaltons. Preferably, each peptide contains at least one unnatural amino acid (i.e., an amino acid that is itself not of the 20 normally found amino acids, or one of the normal 20 amino acids in an abnormal location) and is cyclic in order to protect the peptide from degradation. Although the peptides described herein for the purposes of illustration are separate molecules, the present invention comprehends use of peptides which are attached to one another. In the most preferred embodiment of the invention, the peptide composition includes a combination of cyclic peptides (e.g., peptides having the sequences of Sequence ID Nos. 7, 19, 26, and 34).